The GAN Exonuclease or the Flap Endonuclease Fen1 and RNase HII Are Necessary for Viability of Thermococcus kodakarensis

Abstract

Many aspects of and factors required for DNA replication are conserved across all three domains of life, but there are some significant differences surrounding lagging-strand synthesis. In Archaea, a 5=-to-3= exonuclease, related to both bacterial RecJ and eukaryotic Cdc45, that associates with the replisome specifically through interactions with GINS was identified and designated GAN (for GINS-associated nuclease). Despite the presence of a well-characterized flap endonuclease (Fen1), it was hypothesized that GAN might participate in primer removal during Okazaki fragment maturation, and as a Cdc45 homologue, GAN might also be a structural component of an archaeal CMG (Cdc45, MCM, and GINS) replication complex. We demonstrate here that, individually, either Fen1 or GAN can be deleted, with no discernible effects on viability and growth. However, deletion of both Fen1 and GAN was not possible, consistent with both enzymes catalyzing the same step in primer removal from Okazaki fragments in vivo. RNase HII has also been proposed to participate in primer processing during Okazaki fragment maturation. Strains with both Fen1 and RNase HII deleted grew well. GAN activity is therefore sufficient for viability in the absence of both RNase HII and Fen1, but it was not possible to construct a strain with both RNase HII and GAN deleted. Fen1 alone is therefore insufficient for viability in the absence of both RNase HII and GAN. The ability to delete GAN demonstrates that GAN is not required for the activation or stability of the archaeal MCM replicative helicase. IMPORTANCEThe mechanisms used to remove primer sequences from Okazaki fragments during lagging-strand DNA replication differ in the biological domains. Bacteria use the exonuclease activity of DNA polymerase I, whereas eukaryotes and archaea encode a flap endonuclease (Fen1) that cleaves displaced primer sequences. RNase HII and the GINS-associated exonuclease GAN have also been hypothesized to assist in primer removal in Archaea. Here we demonstrate that in Thermococcus kodakarensis, either Fen1 or GAN activity is sufficient for viability. Furthermore, GAN can support growth in the absence of both Fen1 and RNase HII, but Fen1 and RNase HII are required for viability in the absence of GAN.KEYWORDS Archaea, CMG complex, DNA replication, GINS-associated nuclease GAN, Okazaki fragment, RNase HII, flap endonuclease Fen1 D NA replication is catalyzed by a protein complex, designated the replisome, with structural and catalytic components tightly regulated, intertwined, and coordinated to facilitate both leading-and lagging-strand synthesis. The replisome composition is

The archaeal RecJ-like proteins: nucleases and ex-nucleases with diverse roles in replication and repair

“…The active site of the enzyme is located in the cleft between the DHH and DHHA1 domains, with key residues for substrate binding and catalysis being located in both. The (Fen1) or RNAseHII, both of which likely function in Okazaki fragment processing) is consistent with a role for TkoGAN in this process (12). Note, however, that interactions between RecJ proteins and Pol D have not been reported in other archaeal organisms as yet and so models for TkoGAN function may not translate more widely.…”

“…TK1252 and TK0155, known as TkoGAN and TkoHAN respectively ( Table 1). The TkoGAN protein is perhaps the best characterised RecJ-like protein in the archaea, with information available on activity (9,10), structure (11) and in vivo function (9,12).…”

“…In which case, could the RecJ-like proteins have different roles (catalytic versus structural) depending on whether they are free in solution or part of a CMG, and if so, how can we tease these roles apart? Neither of the models put forward for TkoGAN in Okazaki fragment processing(12) and PfuRecJ in proofreading 3' mismatched primers(22) seems entirely consistent with the exonuclease active site being located in the advancing CMG unwinding DNA at the replication fork. Could these represent non-CMG roles for these enzymes?…”

The small subunit of DNA polymerase D (DP1) associates with GINS–GAN complex of the thermophilic archaea in Thermococcus sp. 4557

“…In most of Euryarchaea including T. kodakarensis , genes encoding both Fen1 and GAN were identified from the genome. Although individual genes can be removed from the genome, the double mutation of GAN plus Fen1 and GAN plus RNAse HII were not successful (Burkhart et al, ). This observation implies that GAN is in a parallel pathway of the known Fen1‐RNAse HII in archaea.…”

Two Archaeal RecJ Nucleases from Methanocaldococcus jannaschii Show Reverse Hydrolysis Polarity: Implication to Their Unique Function in Archaea

“…However, the euryarchaeal Haloferax volcanii did not require the recj gene for its normal growth [ 37 ]. Recent works on T. kodakarensis also demonstrated that GAN could be deleted with no discernable effects on viability and growth, indicating that it is not essential to the archaeal MCM replicative helicase [ 38 ].…”